&RFFROLWKRSKRUHVZRUNVKRSYROXPHObservations on the life cycle and ecology of Acanthoica
quattrospina Lohmann from a Mediterranean estuary.
Luka Šupraha
Department of Earth Sciences, Paleobiology Program, Uppsala University, Villavägen 16, SE-752 36 Uppsala, Sweden;luka.supraha@geo.uu.se

=ULQND/MXEHãLü
Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000, Zagreb, Croatia+UYRMH0LKDQRYLü,QVWLWXWHRI2FHDQRJUDSK\DQG)LVKHULHVäHWDOLåWH,0HåWURYLþD6SOLW&URDWLD

Jorijntje Henderiks
Centre for Ecological and Evolutionary Synthesis, Department of Biosciences, University of Oslo, Blindern PO Box 1066, N-0316 Oslo,
NorwayAbstract: Different life cycle phases of Acanthoica quattrospina life cycle were studied under the scanning electron microscope. Samples were collected during winter 2013 at two stations in the lower reach of the Krka River estuary (Eastern AdriaticSea, Croatia). An ecological analysis revealed that the species was tolerant to low salinity and reached high abundance at the
halocline of the estuary. Examination of holococcoliths showed great variability in the morphology of the central area, indicatingthat several specimens previously described as Sphaerocalyptra sp. represent a variety of the Acanthoica quattrospina holococcolith phase. Furthermore, the observed variability of holococcoliths may represent a distinct mechanism of holococcolith
production, with structural similarities to other members of the Sphaerocalyptra genus.
1. Introduction
Many coccolithophore species exhibit a heteromorphic
life cycle, alternating between morphologically distinct
haploid (holococcolith) and diploid (heterococcolith)
phases (Young et al., 2005). Great progress in understanding their life cycle has been reached by investigations of both spontaneous (Parke and Adams, 1960;
Houdan et al., 2004) and controlled (Nöel et al., 2004)
phase changes in culture material. Furthermore, combination coccospheres (bearing both heterococcoliths andholococcoliths of the same species), that are occasionally
REVHUYHGLQÀHOGVDPSOHVKDYHKHOSHGHVWDEOLVKWKHWD[Rnomic relationships between previously unrelated heterococcolithophore and holococcolithophore taxa (Thomsen
et al., 1991; Cros et al., 2000; Geisen et al., 2002; Triantaphyllou et al., 2004; Malinverno et al., 2008; Frada etal., 2009; Triantaphyllou, 2010).
The family Rhabdosphaeraceae encompasses mostly
morphologically well-documented extant species(Kleijne, 1992; Young et al., 2003). However, only a few
electron micrographs of their holococcolithophore phases
are available to date, including Rhabdosphaera clavigera(Cros and Fortuño, 2002), Algirosphaera robusta (Triantaphyllou and Dimiza, 2003) and Acanthoica quattrospina (Cros et al., 2000; Cros and Fortuño, 2002; Younget al., 2003).
The heterococcolith life phase of A. quattrospina, thetype species of the Acanthoica genus, was described by
Lohmann (1903) and subsequently examined by Schiller(1913). In a detailed review of the Acanthoica genus,
Kleijne (1992) recognized several species having thesame set of coccolith types as A. quattrospina (e.g. A.
acanthifera, A. maxima, A. janchenii) but with different
body coccolith morphology. Most subsequent workers
have only recorded A. quattrospina so it is unclear how
much diversity there is within the genus.

Cros et al. (2000) and Cros and Fortuño (2002) reported
on combination coccospheres containing heterococcolithsof A. quattrospina and previously undescribed holococFROLWKVZLWKDIÀQLW\WRSphaerocalyptra sp. Coccospheres
bearing only holococcoliths that are morphologicallyrelated to ones from combination coccospheres were
found in the same study. However, due to the high degree

Figure 1. Map of the investigated area with two sampling stations E3
and E4a.

50&RFFROLWKRSKRUHVZRUNVKRSYROXPH

Salinity
(PSU)

Total
E. huxleyi
Amount Volume
coccolithoHET
ÀOWHUHG analysed
phores(cells/l)(ml)(ml)(cells/l)

A. quattro- A. quattro- A. quattrospina
spina HET spina COMB
HOL
(cells/l)(cells/l)(cells/l)

Date

StationDepth(m)26.2.2013

E3

0

1.4

100

0.83

7.2 x 103

4.8 x 10300026.2.2013

E3

4

2.5

50

0.41

3 x 105

1.2 x 105

3.6 x 104

7.2 x 103

5.1 x 10426.2.2013

E3

5

22.3

50

0.41

2.6 x 105

1.2 x 105

2.9 x 1040

3.9 x 10426.2.2013

E3

7.5

25.9

50

0.41

3.4 x 105

2.8 x 105

4.8 x 103

2.4 x 103

7.2 x 1035

5

3

26.2.2013

E3

13

27.2

100

0.83

3.1 x 102.8 x 10

3.6 x 100026.2.2013

E3

20

28.8

100

0.83

8.8 x 104

6.5 x 104

2.4 x 1030025.2.2013

E4a

0

4.7

200

1.66

9.7 x 103

7.2 x 103

1.2 x 103

0

0

4

4

3.0 x 107.2 x 1030

1.2 x 10325.2.2013

E4a

3

20.1

100

0.83

5.7 x 1025.2.2013

E4a

5

28.0

200

1.66

5.8 x 104

3.7 x 104

5.4 x 1030

1.8 x 103

1.66

9.8 x 104

4

7.6 x 107.2 x 10

3

0

0

1.66

4

4

2

0

0

25.2.201325.2.2013

E4a
E4a

20
35

28.7
28.8

200
200

2 x 101.7 x 106 x 10

Table 1. Sampling data with the abundance of total coccolithophores and life cycle phases of A. quattrospina.

Figure 2. Plot showing the contribution of each of the life phases vs.
the total abundance of Acanthoica quattrospina at the sampled depths.
Grey area marks the extent of the halocline. Note that the scales for theabundance of total coccolithophores and A. quattrospina are different.

insights into the life cycle of the species, and its particular
ecological preferences.

2 Material and methods
Samples used for this study were collected during winterof morphological variability they could not be unambigu2013 (25-26 February) at two stations in the lower reachously assigned to A. quattrospina, indicating the necessityof the Krka River estuary (Fig. 1). Sampling depths (Table
for more observations on the holococcolith phase of the  ZHUH GHWHUPLQHG DIWHU WKH ÀQH VFDOH H[DPLQDWLRQ RI
species.
temperature, salinity and Chlorophyll a ÁXRUHVFHQFH
During the investigation of coccolithophore assemusing a SeaBird 19plus CTD probe (SeaBird Electronics,
blages of the Krka River estuary (Eastern Adriatic Sea),
Inc. Washington, USA). Seawater for the analysis was
we detected a high abundance of both life cycle phases
obtained simultaneously at all depths, using a series of 5L
of A. quattrospina, as well as combination coccospheres.
Niskin samplers. For quantitative SEM analysis, a known
This allowed us to document the complete morphological
YROXPHRIVHDZDWHU 7DEOH ZDVÀOWHUHGXVLQJDYDFXXP
variability of the holococcolith phase and put our observaSXPSRQWRDPPSRO\FDUERQDWHÀOWHU P&\FORWLRQVLQDQHFRORJLFDOFRQWH[W2XUÀQGLQJVSURYLGHQHZ
SRUH:KDWPDQ $FHOOXORVHQLWUDWHÀOWHURIWKHVDPHVL]H&RFFROLWKRSKRUHVZRUNVKRSYROXPH(0.8 µm Whatman) was placed underneath the polycarERQDWH ÀOWHU WR HQVXUH WKH HYHQ GLVWULEXWLRQ RI PDWHULDO$IWHUÀOWUDWLRQWKHÀOWHUZDVULQVHGZLWKPORIERWWOHG
drinking water (pH=7.54) and dried in the oven at 50°C.
For analysis under a Zeiss Supra35-VP scanning electron
PLFURVFRSH 6(0  D SLHFH RI ÀOWHU ZDV PRXQWHG RQ D
PHWDO VWXE DQG VSXWWHUFRDWHG ZLWK JROG 4XDQWLÀFDWLRQ
of coccolithophores was conducted following the recommendations of Bollmann et al. (2002). Standard taxonomic literature (Cros and Fortuño, 2002; Young et al.,2003) was used for the qualitative analysis of coccolithophore assemblages. For description of the specimens, weused the terminology introduced by Kleijne (1992) and
Young et al. (1997). Since the holococcolithophore phaseof Acanthoica quattrospina had not been described as a
distinct species before the observations on combination51

coccospheres, suitable informal name for the morphotype
is Acanthoica quattrospina HOL, as recommended byYoung et al. (2003).
3. Results
3.1 distribution of A. quattrospina
A sharp halocline was present at both stations, characterized by the increase in salinity from 2.6 to 31.2 PSU
between 3.7-5.3 m depth at the E3 station, and from 6.6 to
36.1 PSU within 1.9-4 m depth at the E4a station (Table
1, Fig. 2). Acanthoica quattrospina was abundant in thistransition layer (up to 9.4 x 104 cells/l) accounting for
maximum 32% of total coccolithophores at the haloclineof the E3 station (Table 1, Fig. 2). Peak abundance atthe E4a station (8.4 x 103 cells/l) was also reached at the
halocline, although with a contribution of only 14% to thetotal coccolithophore count(Table 1, Fig. 2). While
A. quattrospina reachedmaximum abundances at
the halocline, peaks in total
coccolithophore abundance(dominated by Emiliania
huxleyi) were found in themarine layer below the halocline at both stations (Table
1, Fig. 2).
Trends in the vertical
distribution of the heterococcolith and holococcolith
phases differed between thestations. The two life-cycle
phases were equally represented at the halocline of theE3 station, while the holococcolith phase dominateddeeper layers. On the other
hand, station E4a was dominated by the heterococcolith
phase, with minor contributions of the holococcolith phase at the halocline.
Figure 3. SEM micrographs of theheterococcolith phase of Acanthoica quattrospina: A) Typicalcoccosphere of Acanthoica quattropina with two long spines at thebasal end and one long and three
short spines at the apical end (E3,4m). Scale bar=1 µm; B) Acanthoica quattrospina coccospherewith atypical morphology. Note
more than three short apical spinesand high conical protrusions. (E4a,0 m). Scale bar=1 µm; C) Atypical quattrospina morphotype withmany short apical spines. Long
apical spine has fallen off. (E3, 0m). Scale bar= 1 µm.

52&RFFROLWKRSKRUHVZRUNVKRSYROXPH

Combination coccospheres were found only at the halocline and at 7.5 m depth of the E3 station (Table 1, Fig. 2).
3.2 Heterococcolith phase
7KHÀYHVSHFLPHQVRIAcanthoica heterococcolith phase
for which we have high resolution SEM images belonged
to two distinct morphotypes. Three coccospheres exhibited the characteristic morphology of A. quattrospina,

with two long antapical spines and one long spine on theapical end accompanied by three short spines (Fig. 3A).
%RG\UKDEGROLWKVKDGUHODWLYHO\ÁDWFHQWUDODUHDZLWKVKRUWconical protrusion and pronounced cuneate cycle in thecentre. Coccospheres of this morphotype were on average
13.8±2.4 µm long and 10.9±1 µm wide, built of 83-105
rhabdoliths. Two of the observed specimens differed fromthe typical A. quattrospina morphology (Fig. 3B, C),

Figure 4. SEM micrographs of combination coccospheres of Acanthoica quattrospina: A) Complete coccosphere in the heterococcolith phaseshowing two basal spines and one long spine with several short spines on the apical end (E4a, 0 m). Scale bar=1 µm; A) Combination coccosphere
ZLWKIRXUERG\UKDEGROLWKVDQGERG\FDO\SWUROLWKVLQWKHHDUO\VWDJHRIWKHGHYHORSPHQW7ZRFLUFXPÁDJHOODUFDO\SWUROLWKVZLWKOLQHVRIFU\VWDOV
supporting the production of conical protrusion (arrow) are visible (E3, 4 m), Scale bar=1 µm; B) Combination coccosphere with the short apicalspine (arrow) and organic scales that are clearly visible in the centre of the coccosphere (E3, 4 m), Scale bar=1 µm; C) Combination coccosphere
H[KLELWLQJRQHKHWHURFRFFROLWKVKRUWDSLFDOVSLQHDQGWKUHHFLUFXPÁDJHOODUFDO\SWUROLWKV DUURZV  (P 6FDOHEDU P' &RPELQDWLRQFRFFRVSKHUHVKRZLQJIXOO\GHYHORSHGERG\FDO\SWUROLWKVZLWKKLJKFRQHVDQGKROORZFLUFXPÁDJHOODUFDO\SWUROLWK DUURZ  (P 6FDOHEDU P

&RFFROLWKRSKRUHVZRUNVKRSYROXPH

53
Figure 5. SEM micrographs of
A. quattrospina in holococcolith
phase (A-E) and a combination
coccosphere (F): A) Holococcolith
phase showing body calyptroliths
with small conical protrusion (E3,4 m), Scale bar=1 µm; B) Holococcolith phase. Note the building
mechanism of conical protrusion(arrows), where the crystallites
are added laterally onto the skelHWDO VXSSRUWLQJ OLQHV 7KH ÁDJHOODU
opening is visible, surrounded byODUJH FLUFXPÁDJHOODU FDO\SWUROLWKV
(E3, 4 m), Scale bar=1 µm; C) Typical body calyptrolith, tipped with asingle crystallite (E3, 4 m) Scalebar=1 µm; D) Typical holococcoOLWKSKDVHZLWKODUJHFLUFXPÁDJHOODU
calyptroliths (E3, 4m), Scale bar=1µm; E) Dividing coccosphere withfully developed calyptroliths (E3,4m), Scale bar=1 µm; F) Possiblecombination coccosphere showing
the transition from the holococcolith to heterococcolith phase, withshort apical spine (arrow) emerging
DWZKDWVHHPVWREHDÁDJHOODURSHQing (E3, 4m), Scale bar=1 µm.

3.3 Combination
coccospheres
A total of nine combination coccospheres showing
the phase transition wereanalysed during our study(Fig. 4A-D). Some of the
combination coccospheres
still possessed the shortapical spines of A. quattrospina FRQÀUPLQJ WKHLU
WD[RQRPLF DIÀOLDWLRQ )LJ
4B, C). All heterococcoliths in combination coccospheres belonged to thetypical A. quattrospina
morphotype, and no combination coccospheres were found containing rhabdoliths
having more than three short apical spines and body rhabwith the high conical protrusion. Holococcoliths on the
doliths with a high conical protrusion. A high degree ofcombination coccospheres showed different levels ofplasticity was found in body rhabdoliths of the morphostructural complexity and high morphological variability.
type, and the height of their conical protrusion ranged
In the most simple form, only the base of the holococcoIURP D UDWKHU ÁDW FHQWUDO DUHD WR WKH FRQLFDO SURWUXVLRQlith was present, composed of a several crystallites wide
being almost as high as the short apical spines. Average
and one crystallite layer high elliptical ring (Fig. 4A). In
length and width of the coccospheres were 10.4±0.1 µm
some holococcoliths several arches of crystallite units
and 10.1±0.8 µm, respectively. Coccospheres were built
were growing upwards from the edge of the base andof 77-85 rhabdoliths. All analysed specimens possessed
meeting distally (Fig 4A). Finally, most of the holococlong apical and basal spines, typical of the Acanthoica
FROLWKVKDGWKHDUHDEHWZHHQWKHDUFKHVÀOOHGZLWKFU\VWDOgenus, so these atypical specimens may represent an intralites forming a conical protrusion (Fig. 4D, Fig. 5B). As a
VSHFLÀFYDULDQWRIA. quattrospina or a discrete species.
result of the addition of crystallites laterally between thearches, conical protrusions were hollow both on the body

54

&RFFROLWKRSKRUHVZRUNVKRSYROXPH

DQG FLUFXPÁDJHOODU FDO\SWUROLWKV 0LQXWH RUJDQLF ERG\scales were also observed on some of the combination
coccospheres (Fig. 4B).
While most of the combination coccospheres represented the transition from hetero- to holococcolith phase,
we interpret that the coccosphere presented in Fig. 5F
represents the transition from holo- to heterococcolith
phase. The coccosphere possessed only few crystallites
scattered over the unusually small heterococcosphere, on
which body heterococcoliths and one short apical spine
seemed to be emerging from the inside of the cell while
the holococcolith cover was being lost. Furthermore, what
VHHPVWREHWKHÁDJHOODURSHQLQJRIDKRORFRFFROLWKRSKRUH
phase was also visible. The opening was placed where theshort apical spine was emerging, indicating that circumÁDJHOODU KRORFRFFROLWKV GHYHORS DW WKH DSLFDO HQG RI WKHcoccosphere, which corresponds to a pattern observed
on other combination coccospheres (Fig. 4C). However,
WKHÁDJHOODURSHQLQJRIWKHKRORFRFFROLWKSKDVHZDVQRW
clearly observed in our material. A structure resemblingDÁDJHOODURSHQLQJZDVSUHVHQWRQRQO\RQHRIRXUVSHFLmens (Fig 5B).
3.4 Holococcolith phase
A great abundance of cells in the holococcolith phase wasfound in our samples, allowing for the investigation of themorphology of 18 specimens. The spherical coccospheres
were on average 8.4±0.9 µm in diameter, composed of
44-95 holococcoliths. Two main types of holococcoliths
could be distinguished. The body calyptroliths wereÁDWZLWKDEDVDOSODWH RQDYHUDJHPORQJDQG
1.2±0.1µm wide) that was one crystallite layer high, andwith a variable central protrusion (on average 1.1±0.2 µmhigh) tipped with a single crystallite (Fig. 5C). CircumÁDJHOODUFDO\SWUROLWKVKDGDKLJKHUFRQLFDOSURWUXVLRQ RQ
average 1.9±0.3 µm high) ending in an extended tip (Fig.
5B). All crystallites were arranged in a rhomboid pattern,with the base of conical protrusion not reaching the edgeof the calyptroliths base, leaving a basal rim which was
one crystallite high in fully developed calyptroliths (Fig.
5D, E). There was a high degree of variability in themorphology of a central protrusion, which can be rather
small, with a 3-4 crystallites wide rim area (Fig. 5A). In
VRPHVSHFLPHQVLWZDVGLIÀFXOWWRGLVWLQJXLVKERG\FDO\SWUROLWKVIURPFLUFXPÁDJHOODUFDO\SWUROLWKVGXHWRWKHKLJK
central protrusion of body calyptroliths (Fig. 5E).
4. discussion
2XU GDWD SURYLGHV WKH ÀUVW UHSRUW RI KLJK DEXQGDQFH RI
Acanthoica quattrospina in an estuarine coccolithophorecommunity. One of the reasons for high counts in our
samples could be the species’ tolerance to low salinity,
which allowed it to outcompete other coccolithophores in
the unstable environment of the halocline. The haloclineof the Krka River estuary is reported to be the zone of
FRPPRQO\ KLJK QXWULHQW DFFXPXODWLRQ /HJRYLþ et al.,1994) and high bacterial production (Fuks et al., 1991).

Nutrient availability at the halocline could have triggered
the high abundance of A. quattrospina. Furthermore, theSUHVHQFH RI WKH ÁDJHOODWHG KRORFRFFROLWK SKDVH LQ WKHbacteria-rich halocline indicates the potential importanceof phagotrophic nutrition in this species, as was reported
for other coccolithophores (Houdan et al., 2006).
Two distinct heterococcolith Acanthoica morphotypes were detected in our samples, one of which differed
VLJQLÀFDQWO\IURPWKHW\SLFDOA. quattrospina morphology(Fig. 3B-C). Basic body plan and key structural features(locaton of long and short spines and morphology of body
rhabdoliths) suggest that the atypical morphotype may
represent a variant of A. quattrospina. However, only
typical A. quattrospina morphotype was found in combination coccospheres, indicating that there is a genetic andphysiological distinction between the two morphotypes,
and that the atypical morphotype may represent a discrete
species.
Morphological observations provide useful details
on the variability of the holococcolith phase, and help
WR GHWHUPLQH WKH WD[RQRPLFDO DIÀOLDWLRQ RI VSHFLPHQV
SUHVHQWHGE\RWKHUDXWKRUV2XUÀQGLQJVDUHLQDJUHHPHQW
with the micrographs of A. quattrospina HOL found in
SODWHÀJXUHRI<RXQJet al. (2003) and specimens presented on plate VI of Cros et al. (2000). However,
WKHVSHFLPHQVKRZQRQWKHÀJXUHVDQGRISODWHin Young et al.   LGHQWLÀHG DV VS DII A. quattrospina +2/ H[KLELWV VLJQLÀFDQWO\ GLIIHUHQW PRUSKRORJ\of the conical protrusion on body coccoliths compared toour specimens. Cros and Fortuño (2002) reported combination coccospheres containing body rhabdoliths of A.
quattrospina and holococcoliths belonging to Sphaerocalyptra sp. (Fig 18 in Cros and Fortuño 2002). They also
SUHVHQWHGDFRFFRVSKHUHLGHQWLÀHGDVSphaerocalyptra sp.
 )LJ$% ZLWKVLPLODUERG\FRFFROLWKVEXWVLJQLÀcantly higher conical protrusions than the ones found in
the combination coccospheres. Our observations showthat their Sphaerocalyptra sp. 1 represents the holococcolith phase of A. quattrospina, since the variability inthe size of conical protrusion of body coccoliths represents the phenotypic plasticity in this species (both inholococcolith and heterococcolith phase). Furthermore,
FRFFRVSKHUHVRQSODWHÀJXUHRI'LPL]Det al. (2005)
DQGLGHQWLÀHGDVSphaerocalyptra sp.1 show very similarmorphology to A. quattrospina HOL from our samples,with highly variable size of the conical protrusion.
Several specimens of Sphaerocalyptra spp. that werereported in the literature show some morphological similarities with A. quattrospina+2/EXWVWLOOGLIIHUVLJQLÀcantly in key taxonomical characters. Sphaerocalyptra sp.
SUHVHQWHGLQSODWHÀJXUHVRI<RXQJet al. (2003)
has similar body calyptroliths as A. quattrospina HOL,
EXW ZLWKRXW WKH EDVDO ULP DQG FLUFXPÁDJHODU FDO\SWUROiths are much larger than in A. quattrospina HOL, withcrystallites growing spirally towards the top of the conical
SURWUXVLRQ&LUFXPÁDJHOODUKRORFRFFROLWKVRQWKHFRPELQDWLRQFRFFRVSKHUHVKRZQLQÀJXUH$RIRXUPDQXVFULSW

&RFFROLWKRSKRUHVZRUNVKRSYROXPH

have some morphological similarities to SphaerocalyptraVSLOOXVWUDWHGE\&URVDQG)RUWXxR  ÀJXUH
&'LQGLFDWLQJWKDWWKHKRORFRFFROLWKVRQÀJXUH$
could represent a separate form of A. quattrospina HOL.
However, body holococcoliths presented in our work
do not have developed arches, and the extension of theconical protrusion is not as developed as in Sphaerocalyptra sp. 4, suggesting that more observations of that
KRORFRFFROLWKRSKRUH DUH QHHGHG WR SURYH LWV DIÀOLDWLRQ
with A. quattrospina. Another interesting specimen withmorphology similar to A. quattrospina HOL is Sphaerocalyptra cf. adenensisVKRZQLQÀJXUH&'RI&URV
and Fortuño (2002). This specimen has almost cylindrical
FLUFXPÁDJHOODU FRFFROLWKV DQG YHU\ ODUJH FHQWUDO SURWUXsion on body coccoliths lacking the crystallite on top of
the protrusion, different from A. quattrospina HOL.
Since the production of holococcoliths was shownto take place outside of the cell (Nöel et al., 2004), theobserved variability in body calyptroliths could be a
result of incomplete development of a conical protrusion.
An interesting aspect of the morphology of this species
is the apparent mechanism of holococcolith productionthat can be inferred from our observations. We assume
that the production of conical protrusion begins with theinitial construction of several supporting arches meetingin the tip of the cone. Subsequent addition of crystallites
laterally between the arches forms the cone and leaves
the interior of the cone hollow. Such crystallite arches
meeting in the tip of the cone are commonly found inother representatives of the Sphaerocalyptra genus (e.g.
plate 43 of Young et al 2003, plate 1 of Dimiza et al.
2005) and the morphology found in A. quattrospina HOL
seems to be an advanced variant of the same coccolitharchitecture.
A single combination coccosphere that possibly shows
the transition from the holococcolith to the heterococcolith phase was found in our material (Fig. 5F). Previous
investigations of the phase transitions showed that theholococcolith layer is either lost or a simple crystallite
layer is present prior to the production of heterococcoliths, leaving an almost naked cell (Nöel et al., 2004). We
therefore suggest that our specimen may represent theformation of a new heterococcolith phase after the phasetransition.
5. Conclusions
Our work gives a detailed morphological analysis of thelife cycle of Acanthoica quattrospina, with a special focus
on its holococcolith phase and combination coccospheres.
We showed that several previously detected members of
Sphaerocalyptra genus represent the holococcolith phaseof Acanthoica quattrospina. Morphological observations
were put in the ecological context, indicating that species
can reach high abundances in estuarine ecosystems.
Finally, SEM examinations of combination coccospheresare proved to be an important tool for investigations ofcoccolithophore life cycle.

55

Acknowledgements
This research was funded by the Research Council of Norway(FRIMEDBIO project 197823) and the Royal Swedish Academyof Sciences through a grant from the Knut and Alice Wallenberg
Foundation (KAW 2009.0287). We thank the crew and the scienWLÀFVWDIIRQERDUGWKH59+LGUDRIWKH+\GURJUDSKLF,QVWLWXWHRI
WKH5HSXEOLFRI&URDWLDIRUWKHLUKHOSGXULQJWKHÀHOGZRUN:H
are grateful to Jeremy Young, Maria Triantaphyllou, Annelies
Kleijne and Sebastian Meier for reading the manuscript andproviding valuable comments.

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